High-throughput hybridization and reading method for biochips and system thereof

ABSTRACT

A high-throughput hybridization and reading method for biochips uses probes with different marks to specifically connect single nucleotide loci by conducting connection between the probes and target genes at different temperatures, and performing hybridization at the same temperature after the probes are connected, thereby achieving hybridization detection for various loci in a single chip. The method enables fast detection for multiple loci as required by personalized medicine. The detection is high-throughput and systematized and provides highly visualized and highly accurate results. The method allows detection for different loci at different hybridization temperatures to be done simultaneously. The method features highly uniform and repeatable detection, making biochips more efficient and utility in terms of detection. Besides, the chip is easy to prepare and use, thus having a good promotional value.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/182,508, filed Nov. 6, 2018, which claims priority to U.S.provisional application No. 62/739,321 filed Sep. 30, 2018, both ofwhich are incorporated herein.

BACKGROUND OF THE INVENTION 1. Technical Field

The present invention relates to biochip-based assays, and moreparticularly to a high-throughput hybridization and reading method forbiochips and its system.

2. Description of the Related Art

I. Biochips and Gene Chips

A biochip or a bioarray is based on the principle of specificinteractions between biomolecules, and integrates the biochemicalanalysis process in its chip surface, so as to facilitate rapid andhigh-throughput detection of DNA, RNA, polypeptides, proteins and otherbiological components. In a narrow sense, a biochip is a biomoleculelattice made by adhering biomolecules (e.g. oligonucleotides, cDNA,genomic DNA, polypeptides, antibodies, antigens, etc.) to a solid-phasetransmitter, such as a silicon wafer, a glass sheet (bead), a plasticsheet (bead), gel, a nylon film in various ways. As a part of the geneindustry, biochips have great application prospect.

The major features of biochips are high throughput, miniaturization andautomatization. With tens of thousands of closely arranged moleculesintegrated as a microarray, a biochip is capable of analyzing numerousbiomolecules in a short period of time and providing people withaccurate biological information about their samples at a detectionefficiency up to a thousand times of that of the traditional approaches.In the biochip technology, gene chips have been the first commercializedproducts and are currently the most mature. Gene chips are developedbased on the principle of nucleic-acid-probe complementary hybridizationtechnology. The so-called nucleic-acid-probe is just an artificiallycomposed base sequence. The probe is connected to certain detectablesubstance. According to the principle of base complementarity, the geneprobe can be used to identify specific genes in a gene mixture.

On the other hand, a gene chip is also known as a DNA chip or a DNAmicroarray. It is quite similar to chips used in computers, with theonly difference that it has tens of thousands of gene probes denselyintegrated therein as a grid. It uses a DNA fragment having a known basesequence to bond with a single-stranded DNA having a base complementarysequence, so as to determine the corresponding sequence, therebyidentifying abnormal genes or their products. Currently, gene chips fordetection of gene mutation and gene expression spectrum chip fordetection of gene expression levels are relatively mature products. Thegene chip technology principally comprises four fundamental technicalparts: preparation of chip microarrays, preparation of samples,biomolecule reaction, as well as detection and analysis of signals.

II. Biochips for SNP detection

In 2001, the SNP Consortium developed variation human genetic mapcontaining 1.42×10⁶ SNPs, with a density of one single-nucleotidepolymorphism locus in every 1.9 kb-fragment. This is every important toresearches on virulence genes and the origin and evolution of mankind.As estimated, there will be about one hundred thousand SNP molecularmarkers used for researches on gene functions and diseases. Such hugeanalysis needs demand highly for detection technologies.

At present, there are several common methods for detection ofsingle-nucleotide polymorphism of alleles.

The first is allele-specific amplification. It is to make the 3′-end ofthe designed Primer 1 complementary to the sequence of a to-be-detectedP allele in a polymerase chain-reaction system, and not complementary tothe sequence of another allele (Q). During the PCR amplification, the Pallele amplified. In the same manner, the designed Primer 2 iscomplementary to the sequence of Q, and then the amplified products areanalyzed through electrophoresis respectively. While this known methodcan detect single-nucleotide polymorphism, its operation is complicatedand it has higher requirements about primer design, resulting indifficult operation and huge workload.

The second is bidirectional PCR amplification of specific alleles(bi-PASA), which is based on the same principle as allele-specificamplification except that bi-PASA uses 4 primers, wherein Primer F1 hasits sequence complementary to that of the 5′-end in a to-be-detected Xallele; Primer R2 has its sequence complementary to that of the 3′-endin a to-be-detected Y allele; Primer R1 is located middle-to-downstreamin the sequence of X, with its 3′-end being X; and Primer F2 is locatedmiddle-to-upstream in the sequence of Y, with its 3′-end being Y.Therein, X and Y each represents a polymorphic locus. During PCRamplification, two oligonucleotide sequences containingsingle-nucleotide polymorphism loci can be obtained simultaneously. Thisallows a target gene to be identified in a single round of polymerasechain reaction. While this known approach is operationally simple andeconomic, it is conducted in the liquid phase and there is only alimited number of multiple parallel reactions, thus failing to providehigh-throughput detection for single-nucleotide polymorphism.

The third is primer extension, which identifies base polymorphism lociusing DNA polymerase, and provides better specificity as compared tothose methods based on allele-specific hybridization. This kind ofmethods have many names, such as minisequencing, single-nucleotideprimer extension (SnuPE), primer-guided nucleotide incorporation, andtemplate directed dye terminator incorporation (TDI). Therein,minisequencing is the most popular. The process involves obtaining afragment of DNA containing single-nucleotide polymorphism loci firstthrough amplification, and then performing minisequencing, in which adetection primer is introduced, its 3′-end base is immediately next tothe polymorphism base, and extension of a base is conducted with thepresence of DNA polymerase and marked ddNTPs. The extended base is apolymorphism base. Since the primer for the polymerase chain reactioncompetes with the extension primer, plural amplified fragments aregenerated, the surplus dNTPs lead to extension of plural bases in theextension reaction. Thus, before minisequencing, the polymerase chainreaction products have to be purified in order to remove the polymerasechain reaction primers and dNTPs contained therein. The primer extensionproducts may be detected using radioisotope labeling, luminescencedetection, gel-based fluorescence detection, mass spectrometry, anddenaturing high performance liquid chromatography. However, thesemethods more or less have disadvantages and shortcomings about highcosts and complicated operation. For example, while radioisotopelabeling and luminescence are highly automatized and convenient, theyrequire high equipment costs. On the other hand, gel-based fluorescencedetection is relatively economic yet requires complicated andeffort-consuming preparation.

Analysis of single-nucleotide polymorphism based on allele-specificoligonucleotide (ASO) hybridization is the simplest hybridization-baseddetection method. During hybridization of a short nucleic-acid probe anda complementary target fragment, the hybridization complex will havedifferent levels of stability for a perfect match case and a mismatchcase, and single-nucleotide polymorphism loci can be detected based onthis difference. This method first involves designing a shortnucleic-acid probe, normally 15-20 bp. The probe containssingle-nucleotide polymorphism loci. When it is hybridized with a sampleDNA, a one-base difference in the 20 bp results in a Tm value decreaseof 5-7.5 degrees. Thus, by strictly controlling the conditions forhybridization, it is possible to determine whether there issingle-nucleotide polymorphism in the sample DNA. However, the defectsof this method lie on not only the difficulty in stringently controllinghybridization, but also incapability of distinguishing single-nucleotidepolymorphism loci more accurately.

To realize more stringent hybridization and to better distinguishsingle-nucleotide polymorphism loci, a modified nucleic acid probe isused for hybridization with the sample DNA, such as a peptide nucleicacid (PNA) probe. Nevertheless, a probe made of peptide nucleic acid isless soluble, thus being unfavorable to reaction. Besides, the probeshall be at least as long as 7 bases, so as to ensure good hybridizationat room temperature. For a probe rich in guanine rhodamine markers, itshigh background fluorescence polarization (FP) signals also poseproblems. Another reported solution is to artificially insert a mismatchbase (3′-nitropyrrole) into a probe. By using such a probe containingmismatch bases for hybridization, the one-base difference between theprobe and a target fragment can decrease the Tm value twice as many asusing the traditional hybridization, making hybridization more specific.However, this known method requires strict control on hybridizationconditions, and its detection is not accurate enough.

TagMan is a method for in vitro gene amplification based on theprinciple of hybridization and conventional polymerase chain reaction.The existing method involves adding probes designed forsingle-nucleotide polymorphism loci and flanking sequences into apolymerase chain reaction system. There is only a one-base differencebetween different probes, corresponding to different alleles,respectively. In addition, the probes are marked with fluorescence andare terminally phosphorylated to prevent the probe from being extendedduring amplification. The probes can specifically bond with the targetsequence. When the probe is intact, due to the quenching group, thefluorescent marker is prevented from fluorescence through fluorescenceresonance energy transfer (FRET). During amplification in polymerasechain reaction, with the activity of the exonucleases of the TagMan DNApolymerase 5′-3′-end, the fluorescent marker at the 5′-end of the probecan be cut off from the probe, thereby allowing fluorescence signal tobe released. Since the process happens during polymerase chain reaction,neither isolation nor elution is required, thereby reducing the risk ofPCR contamination. Besides, analysis of the amplified products isconvenient, fast, and accurate, without using electrophoresis, so theexperiment can be done more rapidly. However, since the designed probeshave only one-base differences, the requirement is very strict.Moreover, the detection result is merely useful in determination ofgenotype of one SNP locus, making the detection throughput limited andfailing to meet the current demand of high-throughput detection.

CN 101034061 discloses a method for detecting single-nucleotidepolymorphism with biochips, which involves performing amplification on anucleic acid sample first to obtain an amplified product; thenperforming liquid phase hybridization and ligase reaction to connect theperfect match probe in solution hybridization with the universal probe,thereby detecting single nucleotide polymorphism; and performingsolid-phase hybridization to hybridize the probe having label probes atits 5′-end and detection groups at its 3′-end with the biochip at 25°C.-75° C. for 0.5 h-36 h, so that the detection groups are distributedto different sites in the biochip; and performing single-nucleotidepolymorphism detection to get results. However, the patented method doesnot teach how to make detection of multiple single-nucleotidepolymorphism loci in a high-throughput manner.

The prior art also has the following shortcomings:

1. The detection adds workload and is time-consuming. The conventionalsingle-nucleotide polymorphism detection methods such as normalpolymerase chain reaction and fluorescence quantitative polymerase chainreaction can only detect one or a few single-nucleotide polymorphismlocus at a time, and are incapable of simultaneous detection fornumerous single-nucleotide polymorphism loci;2. The probes provided in the traditional biochips are allspecies-specific, so each chip is only useful in detection of a singleparticular species. For change or for addition of the detection objects,the only way is to make a new chip. Thus, the prior art is inefficientand not suitable for simultaneous detection of multiple samples anddetection of plural loci in a single sample.

SUMMARY OF THE INVENTION

In view of the shortcomings of the prior art, the present inventionprovides a high-throughput hybridization and reading method forbiochips, which simultaneously detects plural SNP loci in a single chip.For detection of each SNP locus, the method comprising the followingsteps:

a) performing PCR amplification on a double-stranded DNA to be detected,and marking one of its strands with a marker;

b) separating the amplified double-stranded DNA, keeping the DNA strandlabeled with the marker, and naming it as a to-be-detected DNA strand;

c) introducing the to-be-detected DNA strand, a first probe, a secondprobe, and a third probe into one reaction system for polymerase chainreaction, wherein, the 3′-end through 5′-end of the first probe are afirst hybridization region and a first complementary region,respectively, and the 5′-end is an A base or a G base; the 3′-endthrough 5′-end of the second probe are a second hybridization region anda second complementary region, respectively, and the 5′-end is a C baseor a T base; the 5′-end of the third probe is attached to fluorescentgroups or chromophoric groups; and neither of the first and the secondhybridization regions is complementary to the to-be-detected DNA,both of the first and the second complementarity regions arecomplementary to the to-be-detected DNA strand, the 5′-end of the secondprobe or the first probe is complementary to an SNP locus of theto-be-detected DNA, the first probe and the second probe have at least10 same bases from their 5′-ends to the 3′-ends and have differentintervals, which comprise at least 50% of all bases of each probe; andd) when the polymerase chain reaction ends, transferring the reactionliquid to the biochip, which has been fixed with fragments complementaryto the first hybridization region of the first probe and the secondhybridization region of the second probe, respectively, therebyaccomplishing hybridization of the single SNP locus;For detection of multiple SNP loci, depending on SNP loci of thebe-detected DNA, designing a first, a second, and a third probesequences, and repeating Step a) through Step d); so as to obtain a chipor chips having the plural SNP loci;performing specific reaction with the resulting chip(s) to obtainprecipitation or fluorescence, and determining types of the SNP lociaccording to results of the specific reaction.

According to a preferred mode, the marker in Step a) includes but is notlimited to: a biotin, an avidin, and a streptavidin.

According to a preferred mode, the fluorescent group of the third probein Step c) includes but is not limited to: FAM, HEX, TET, JOE, TAMRA,Texas Red, ROX, CY3 and CY5.

According to a preferred mode, the chromophoric group of the third probein Step c) includes but is not limited to: ECL, NBT/BCIP and DAB.

According to a preferred mode, the number of the bases in each of thefirst and second hybridization regions is 15 to 25.

According to a preferred mode, the number of the bases in each of thefirst and second complementarity regions is 15 to 25.

According to a preferred mode, detection channels for the fluorescentgroups are: FAM; 465 to 510 nm; CY3: 533 to 580 nm; HEX: 533 to 580 nm;TET: 533 to 580 nm; JOE: 533 to 580 nm; Texas Red: 533 to 610 nm; ROX:533 to 610 nm; CY5: 618 to 660 nm; and TAMRA: 533 to 580 nm.

According to a preferred mode, Tm values corresponding to the probes areindependently greater than 25° C.

According to a preferred mode, PCR amplification system is a 25 μLreaction system, which comprises: 10×PCR buffer solution 2 μL, 25 mMMgCl 1.5 μL, 0.2 mM×dNTPs 0.5 μL, 5 U/μL Taq DNA polymerase 0.25 μL, 100μM forward primer 0.1 μL, 100 μM reverse primer 0.1 μL, ddH20 making upto 25 μL

According to a preferred mode, the reaction condition for the PCRpolymerase chain reaction is: 95° C. initial denaturation 5 min; 95° C.2 s; 58° C. 10 s; 60° C. 1 min, 40 cycles in total.

The present invention further provides a high-throughput hybridizationand reading system for biochips. The system uses the disclosedhigh-throughput hybridization and reading method for biochips. Thehigh-throughput hybridization and reading system for biochips issuitable for SNP detection of various types, such as bioassay, clinicalmedicine detection, prediction and diagnosis of catastrophic diseases,plotting of genetic maps, animal and plant inspection and quarantine,and so on.

The high-throughput hybridization and reading system for biochips cansimultaneously detects plural SNP loci in a single chip. The systemcomprises:

an amplification unit, for a) performing PCR amplification on adouble-stranded DNA, and marking one of its strands with a marker;

a separation unit, for b) separating the amplified double-stranded DNA,keeping the DNA strand labeled with the marker, and naming it as ato-be-detected DNA strand;

a polymerase chain reaction unit, for c) introducing the to-be-detectedDNA strand, a first probe, a second probe, and a third probe into onereaction system for polymerase chain reaction, wherein, the 3′-endthrough 5′-end of the first probe are a first hybridization region and afirst complementary region, respectively, and the 5′-end is an A base ora G base; the 3′-end through 5′-end of the second probe are a secondhybridization region and a second complementary region, respectively,and the 5′-end is a C base or a T base; the 5′-end of the third probe isattached to fluorescent groups or chromophoric groups; and: neither ofthe first and the second hybridization regions is complementary to theto-be-detected DNA, both of the first and the second complementaryregions are complementary to the to-be-detected DNA strand, the 5′-endof the second or the first probe is complementary to an SNP locus of theto-be-detected DNA, the first probe and the second probe have at least10 same bases from their 5′-ends to the 3′-ends and have differentintervals, which comprise at least 50% of all bases of each probe; anda chip hybridization detection unit, for d) when the polymerase chainreaction ends, transferring the reaction liquid to the biochip, whichhas been fixed with fragments complementary to the first hybridizationregion of the first probe and the second hybridization region of thesecond probe, respectively, thereby accomplishing hybridization of thesingle SNP locus;for detection of multiple SNP loci, depending on SNP loci of thebe-detected DNA, designing a first, a second, and a third probesequences, and repeating Step a) through Step d), so as to obtain a chipor chips having the plural SNP loci; andperforming specific reaction with the resulting chip(s) to obtainprecipitation or fluorescence, and determining types of the SNP lociaccording to results of the specific reaction.

According to a preferred mode, the marker in Operation a) includes butis not limited to: a biotin, an avidin, and a streptavidin.

According to a preferred mode, the fluorescent group of the third probein Operation c) includes but is not limited to: FAM, HEX, TET, JOE,TAMRA, Texas Red, ROX, CY3 and CY5.

According to a preferred mode, the surface of the biochip is fixed witha large number of label-complementary probes corresponding to differentsingle-nucleotide polymorphism loci.

The present invention has one or more of the following beneficialeffects:

As compared to the prior art, the disclosed high-throughputhybridization and reading method for biochips uses probes with differentmarks to specifically connect single nucleotide loci, the probes areconnected with target genes at different temperatures, hybridizationsare performed at the same temperature after the probes are connected,thereby achieving hybridization detection for various loci in a singlechip. The present invention adopts digoxin markers or fluorescentmarkers to address the problem with the Taqman probes that are highlytemperature-dependent, and can only detect a single-nucleotide locus atone time. With the three different probes to match to-be-detected SNPlocus, the present invention allows parallel detection of different SNPlocus, and can accurately read information of SNP locus from the chips.The detection of samples and SNP locus is sensitive and high-throughput,while significantly reduces detection costs and use of fluorescenceprobes. Moreover, the detection results are highly repeatable anduniform. The method supports parallel tests and readings in just a fewsteps, so the operation is simple and convenient. Besides, the chip iseasy to prepare and operate, so the method is worthy of promotion.

According to the present invention, a large number of differentlabel-complementary probes are fixed on the surface of biochips,different label complementary probes corresponding to differentsingle-nucleotide polymorphism locus, therefore this method cansimultaneously detect more than one hundred different single-nucleotidepolymorphism loci.

According to the present invention, the label-complementary probes inthe biochip have a relatively high melting point, and hybridizationbetween the label-complementary probe and the label probe is highlyspecific, so the time required by detection can be significantlydecreased.

The present invention provides an open system because the probes in thebiochip are label-complementary and independent of the detectionobjects. By simply changing the probes in the solution hybridizationsystem, detection of different detection objects can be achieved withoutchanging the probes in the biochip. Thus, the biochip of the presentinvention is applicable to detection of various single-nucleotidepolymorphism variations. The present invention is convenient, fast,sensitive, and specific, thus having extensive applications. The presentinvention can be used to detect trace nucleic-acid samples, can identifygenotypes among tens of thousands of highly parallel polymorphismvariations, and can be used in identifying pathogenic microorganism,disease diagnosis, locating pathogenic loci, quantitative trait locusanalysis, identifying loss of heterozygosity of cancer genes and relatedresearches.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the principle of hybridization and reading accordingto the present invention; and

FIG. 2 is a biochip hybridization spectrum of an experiment according toan embodiment of the present invention.

DETAILED DESCRIPTION

The invention as well as a preferred mode of use, further objectives andadvantages thereof will be best understood by reference to the followingdetailed description of illustrative embodiments when read inconjunction with the accompanying drawings.

Unless stated otherwise, the experiment methods used in the followingembodiments are all those conventional. Unless stated otherwise, theexperiment materials used in the following embodiments are allcommercially available.

Embodiment 1

I. Probe Design

In the present invention, different probes correspond to differentsingle-nucleotide loci. The present invention places no particularlimitations on the location of different single-nucleotide loci in agenomic DNA. The different probes are designed according toto-be-detected single-nucleotide loci. The present invention places noparticular limitations on how the probe is designed, and the design canbe done using any probe design principles and software known to peopleskilled of the art. For example, multiple sequence alignment softwaresuch as Clustal may be used to do multiple sequence alignment, and LSPDsoftware may be used to design probes and primers.

In the present embodiment, the to-be-detected SNP loci are two SNP lociin an APOE gene, located in Locus 112 and Locus 158. The sequence is asdescribed in SEQ ID NO: 1 and is shown below, wherein the to-be-detectedSNP loci are bold and underlined:

ggcacggctgtccaaggagctgcaggcggcgcaggcccggctgggcgcg gacatggaggacgtg tgcggccgcctggtgcagtaccgcggcgaggtgcaggccatgctcggccagagcaccgaggagctgcgggtgcgcctcgcctcccacctgcgcaagctgcgtaagcggctcctccgcgatgccgatgacctg cagaag cgcctggcagtgtaccaggccggggcccgcgagggcgccgagc gcggcctcagcgccatccgcga

As shown in FIG. 1, the first probe 1 and the second probe 2 aredesigned according to SNP loci of the to-be-detected fragment. Therein,the first probe 1 and the second probe 2 each have a length of 30 bp.The first probe 1 has a first hybridization region and a firstcomplementary region from its 3′-end, and its 5′-end is an Abase. The3′-end through 5′-end of the second probe are a second hybridizationregion and a second complementary region, respectively, and the 5′-endis a C base. The first probe and the second probe are different in allthe 12 bases in their hybridization regions, yet in the complementarityregions, their 17 bases are the same and match the amplified products.This ensures that the first probe and the second probe have at least 10same bases from their 5′-end to 3′-end and have different intervals,which comprise at least 50% of all bases of each probe. The 5′-end ofthe third probe is attached to fluorescent groups or chromophoricgroups. The complementary region of the second probe and the first probehybridize with the to-be-detected fragment. The spotting probe iscomplementary to the hybridization region of the first or the secondprobe. The third probe and the first probe are complementary to theto-be-detected fragment at two sides of the to-be-detected SNP locus.After match, with the effect of Taq polymerase, a complete probe strandis formed.

According to the foregoing probe design rules, the probes of the presentembodiment are as shown in Table 1 below:

TABLE 1 Locus Probe SEQ ID NO. DNA Sequence (3′-5′) APOE First Probe 1 4 gcggtagtaccatacggtacctcctgcaca Locus 112 Second Probe 2  5acttggtctagccgacctacctcctgcacc Spotting Probe 1  6 cgccatcatggtatgccSpotting Probe 2  7 tgccacagatcggctgg Chromogenic  8cgccggcggaccacgtcatgg Probe 5 APOE First Probe 3  9cgtcatgtgcaatccgactggacgtcttcg Locus 158 Second Probe 4 10atggagcattccgaacgctggacgtcttct Spotting Probe 3 11 gcagtacacgttaggctSpotting Probe 4 12 tacctcgcggattcgac Chromogenic 13cggaccgtcacatggtccggc Probe 6

In the present embodiment, the probes match the to-be-detected fragmentfrom its 5′-end. In other words, the first probe has from its 3′-end thefirst hybridization region and the first complementary regionsuccessively, and the 5′-end is an A base or G base. The 3′-end through5′-end of the second probe are the second hybridization region and thesecond complementary region, respectively, and the 5′-end is a base C orT. The 5′-end of the third probe is connected with fluorescent groups orchromophoric groups. In a different design scheme, the probes match theto-be-detected fragment from its 3′-end instead. In other words, thefirst probe has from its 5′-end the first hybridization region and thefirst complementary region successively, and the 3′-end is an A base ora G base. The second probe has from its 5′-end the second hybridizationregion and the second complementarity region successively, and the3′-end is a base C or T. The third probe has is 5′-end phosphorylated,and has its 3′-end connected with fluorescent groups or chromophoricgroups. This forms a design directionally opposite to the presentembodiment, yet similarly achieving match and hybridization.

II. Double-Stranded PCR Amplification

PCR double-stranded amplification is performed on the to-be-detectedfragment, and one strand of the amplified product is connected withbiotin microspheres to facilitate strand separation.

Primers are designed as below according to the to-be-amplified fragment:

ApoE-primer-1 (as described in SEQ ID NO: 2): tcgcggatggcgctgaApoE-primer-2 (as described in SEQ ID NO: 3): biotin-ggcacggctgtccaaggaTherein, one end of Primer 2 is connected with biotin microspheres.

The PCR amplification system is a 250 μL reaction system, whichcomprises: 10×PCR buffer solution 2 μL, 25 mM MgCl 1.5 μL, 0.2 mM×dNTPs0.5 μL, 5 U/μL Taq DNA polymerase 0.25 μL, 100 μM forward primer 0.1 μL,100 μL, 100 μM reverse primer 0.1 μL, and ddH20 making up to 25 μL.Therein, the 5′-end of the forward primer is connected with biotinlabel.

The PCR polymerase chain reaction is performed with the followingconditions: 95° C. initial denaturation 5 min; 95° C. 2 s; 58° C. 10 s;60° C. 1 min, 40 cycles in total.

In the present invention, the forward primer and reverse primer areprimers designed according to the to-be-detected SNP locus. The presentinvention places no particular limitations on how the primers aredesigned, and the design may be done using any primer design methodknown to people skilled of the art. The present invention places noparticular limitations on the conditions of the polymerase chainreaction, and conventional PCR reaction conditions known to peopleskilled of the art may be used.

III. DNA Strand Separation

DNA strand separation may be realized by filtration separation using aDNA strand separation device, or using other known methods. The DNAstrand separation device uses polyethylene microspheres as its membranefilter material, with gaps between the microspheres preferably being 10μm, smaller than the diameter of the biotin, so that by direct physicalfiltration, single strands with affinity linker can be kept on themembrane, the strands without the affinity linker are filtered off. Thedevice provides good adsorption and elution, and has a high DNA recoveryrate. Besides, the material is inexpensive and environmentally friendly.

The separation process involves: adding 0.4M NaOH and 1M NaCl to unwindthe double stranded helix, gently blowing and agitating forhomogenization, centrifuging at 12000 rmp for 1 min; washing offresidual NaOH, neutralizing to neutrality in pH, centrifuging at 12000rmp for 1 min; with the to-be-detected fragment retained by the membranefilter, adding a collecting liquid such as ultrapure water, then gentlyblowing and agitating until the DNA strand fully suspend, collecting thestrand, sealing up and storing at 4° C. for later use.

IV. Integrated Reaction for Hybridization and Reading

Hybridization of the first probes 1, 3 and the second probes 2, 4 andthe third probes 5, 6 with the single-stranded DNA is performed using aDNA ligase. The hybridization system works with the followingconditions: 95° C. 30 s, 60° C. 30 s, 72° C. 30 s, 35 cycles in total.For hybridization, the DNA ligase may be T4 DNA ligase or a thermostableDNA ligase. For better ligation, a thermostable DNA ligase is preferred.The buffer solution used in the hybridization system may be formulatedsimilarly as to that for amplification.

The chromogenic probes are diluted to 5 pmol/μl. The first probe or thesecond probe has a concentration of 0.25 to 1 pmol/μl. For preparing thehybridization solution, 1 μl of the first probe or the second probe, and1 μl of the chromogenic probe are added into 250 μl hybridizationbuffer. The resulting hybridization solution and the amplified productsgo through hybridization reaction together.

After hybridization, a hybrid “amplified product—first probe 1—thirdprobe 5” and a hybrid “amplified product—first probe 3—third probe 6”are formed. The hybrids are eluted and neutralized to pH7-8 using theeluate for chip hybridization detection.

A spotting probe 1 matching the first probe 1 and a spotting probe 2matching the second probe 2 are fixed to the chip. The spotting probesare diluted to 10 pmol/ul, and spot on the chip in order.

The sequence of the spotting probe 1 is complementary to the firsthybridization region of the first probe 1, and the sequence of thespotting probe 2 is complementary to the second hybridization region ofthe second probe 2. The first probe 1 and the second probe 2 arehybridized with the sequences fixed to the chip, respectively. Afterhybridization, the identification fragment at the end of the first probeor the second probe is used to perform SNP locus variant identification.The chip is generally made of a nylon membrane, while a glass sheet or asilicone sheet is also usable

After the reaction, the reactant is washed, and a digoxin antibody isadded for further reaction. After the further reaction, the reactant iswashed again. Then a chromogenic solution is added for chromogenicreaction. Hybridization, antibody reaction, and chromogenic reactioneach take 10 minutes.

Since the signal probe exclusively has perfect match with a singlegenotype of the SNP locus, the type of the bases of the to-be-detectedSNP locus can be identified according to chromogenic result of the chip.

After hybridization of the probes and the amplified products,centrifuging is performed to remove unbound parts, and a digoxinantibody is added to react with the digoxin at the end of the hybridizedthird probe. After the reaction, the antibody is washed off, andcatalysis is performed using BCIP/NBT to form blue-violet precipitationon the surface of the chip.

Where the chromogenic probe is terminated with fluorescent groups, theforegoing antibody reaction can be eliminated and fluorescence atdifferent sites in the chip can be directly observed.

In the present embodiment, at Locus 112, the first probe 1 and thechromogenic probe 3 perfectly match the amplified products. At Locus158, the first probe 3 and the chromogenic probe 6 perfectly match theamplified products. As shown in FIG. 2, the first column represents thespotting probe 1, the second column represents the spotting probe 2, thethird column represents the spotting probe 3, and the fourth columnrepresents the spotting probe 4. During hybridization of chips, only thefirst probe 1 and the first probe 3 are left in the reaction system tomatch the spotting probes in the chip. Therefore, the first column andthe fourth column form blue-violet precipitation on the surface of thechip. This further shows that the base sequence at Locus 112 is T, andthe base sequence at Locus 158 is C.

In the present embodiment, each of the first and second probes is madewith three different concentrations (0.25 pmol/μL, 0.5 pmol/μL, and 1pmol/μL). Each concentration corresponds to two sets of parallel tests,amounting to 6 sets. The results of the parallel tests are shown inFIG. 1. Precipitation is formed at the same probes.

The disclosed hybridization and reading system can accomplishsimultaneous parallel analysis of 24 samples in merely 60 minutes. Thedetection results are consistent and the operation is convenient yeteffective. The precipitation results are captured using external camerasand the images are automatically processed so that the signal pointsshowing after hybridization chromogenic reaction are marked. Afterhybridization, the chromogenic location of reaction sites in the chip orthe chromogenic intensity can be analyzed using a chip scanner andrelated software, and the imaging signals can be converted into data toprovide the related biological information, thereby rapidly completingthe entire operation from the raw samples to the desired analysisresults in a closed system in a short period of time.

More conditions and parameters for the disclosed hybridization andreading process claimed herein can be seen from the existing methods andsystems for SNP detection using biochips. The present embodiment isfocused on the characteristics of the disclosed method and thedifference between the present invention and prior art, and omitsunnecessary details that are known to people skilled in the art.

Embodiment 2

The present embodiment is similar to Embodiment 1 with the onlydifference that fluorescence is used for chip reading. The results areconsistent with Embodiment 1.

The present invention has been described with reference to the preferredembodiments and it is understood that the embodiments are not intendedto limit the scope of the present invention. Moreover, as the contentsdisclosed herein should be readily understood and can be implemented bya person skilled in the art, all equivalent changes or modificationswhich do not depart from the concept of the present invention should beencompassed by the appended claims.

What is claimed is:
 1. A high-throughput hybridization and readingmethod for biochips, which simultaneously detects plural SNP loci in asingle chip, for detection of each SNP locus the method comprising thefollowing steps: a) performing PCR amplification on a double-strandedDNA to be detected, and marking one of its strands with a marker; b)separating the amplified double-stranded DNA, keeping the DNA strandlabeled with the marker; c) introducing the separated amplifiedto-be-detected DNA strand, a first probe, a second probe, and a thirdprobe into one reaction system for polymerase chain reaction, wherein,the 3′-end through 5′-end of the first probe are a first hybridizationregion and a first complementary region, respectively, and the 5′-end isan Abase or a G base; the 3′-end through 5′-end of the second probe area second hybridization region and a second complementary region,respectively, and the 5′-end is a C base or a T base; and the 5′-end ofthe third probe is attached to fluorescent groups or chromophoricgroups; and: neither of the first and the second hybridization regionsis complementary to the to-be-detected DNA, both of the first and thesecond complementary regions are complementary to the separatedamplified to-be-detected DNA strand, the 5′-end of the second probe orthe first probe is complementary to an SNP locus of the to-be-detectedDNA, the first probe and the second probe have at least 10 same basesfrom their 5′-ends to the 3′-ends and have different intervals, whichcomprise at least 50% of all bases of each probe; d) when the polymerasechain reaction ends, transferring the reaction liquid to the singlebiochip, which has been fixed with fragments complementary to the firsthybridization region of the first probe and the second hybridizationregion of the second probe, respectively, thereby accomplishinghybridization of the single SNP locus; designing a first, a second, anda third probe sequences as a function of SNP loci of the separatedamplified be-detected DNA, and repeating Step a) through Step d), so asto obtain the single chip for detecting the plural SNP loci; andperforming specific reaction with the resulting single chip to obtainprecipitation or fluorescence, and determining types of the SNP lociaccording to results of the specific reaction.
 2. The method of claim 1,wherein the marker in Step a) includes but is not limited to: a biotin,an avidin or a streptavidin.
 3. The method of claim 1, wherein thefluorescent group of the third probe in Step c) includes but is notlimited to: FAM, HEX, TET, JOE, TAMRA, Texas Red, ROX, CY3 and/or CY5.4. The method of claim 1, wherein the chromophoric group of the thirdprobe in Step c) includes but is not limited to: ECL, NBT/BCIP or DAB.5. The method of claim 1, wherein the number of the bases in each of thefirst and second hybridization regions is 15 to
 25. 6. The method ofclaim 1, wherein the number of the bases in each of the first and secondcomplementary regions is 15 to
 25. 7. The method of claim 1, whereindetection channels for the fluorescent groups are: FAM: 465 to 510 nm;CY3: 533 to 580 nm; HEX: 533 to 580 nm; TET:533 to 580 nm; JOE: 533 to580 nm; Texas Red: 533 to 610 nm; ROX: 533 to 610 nm; CY5: 618 to 660nm; and TAMRA:533 to 580 nm.
 8. The method of claim 1, wherein Tm valuescorresponding to the probes are independently greater than 25° C.
 9. Themethod of claim 1, wherein the PCR amplification system is a 25 μLreaction system, which comprises: 10×PCR buffer solution 2 μL, 25 mMMgCl 1.5 μL, 0.2 mM×dNTPs 0.5 μL, 5 U/μLTaq DNA polymerase 0.25 μL, 100μM forward primer 0.1 μL, 100 μM reverse primer 0.1 μL, ddH20 making upto 25 μL.
 10. The method of claim 1, wherein the reaction condition forthe PCR polymerase chain reaction is: 95° C. initial denaturation 5 min;95° C. 2 s; 58° C. 10 s; 60° C. 1 min, 40 cycles in total.